Remberg A, Ruddat A, Braslavsky S E, Gärtner W, Schaffner K
Max-Planck-Institut für Strahlenchemie, P.O. Box 101365, D-45413 Mülheim an der Ruhr, Germany.
Biochemistry. 1998 Jul 14;37(28):9983-90. doi: 10.1021/bi980575x.
N-Terminal apoprotein fragments of oat phytochrome A (phyA) of 65 kDa (amino acids 1-595) and potato phyB of 66 kDa (1-596) were heterologously expressed in Escherichia coli and in the yeasts Saccharomyces cerevisiae and Pichia pastoris, and assembled with phytochromobilin (PthetaB; native chromophore) and phycocyanobilin (PCB). The phyA65 apoprotein from yeast showed a monoexponential assembly kinetics after an initial steep rise, whereas the corresponding apoprotein from E. coli showed only a slow monoexponential assembly. The phyB66 apoprotein incorporated either chromophore more slowly than the phyA65s, with biexponential kinetics. With all apoproteins, PthetaB was incorporated faster than PCB. The thermal stabilities of the Pfr forms of the N-terminal halves are similar to those known for the full-length recombinant phytochromes: oat phyA65 Pfr is highly stable, whereas potato phyB66 Pfr is rapidly converted into Pr. Thus, neither the C-terminal domain nor homodimer formation regulates this property. Rather, it is a characteristic of the phytochrome indicating its origin from mono- or dicots. The Pr to Pfr kinetics of the N-terminal phyA65 and phyB66 are different. The primary photoproduct I700 of phyA65-PCB decayed monoexponentially and the PthetaB analogue biexponentially, whereas the phyB66 I700 decayed monoexponentially irrespective of the chromophore incorporated. The formation of Pfr from Pr is faster with the N-terminal halves than with the full-length phytochromes, indicating an involvement of the C-terminal domain in the relatively slow protein conformational changes.
燕麦光敏色素A（phyA）65 kDa（氨基酸1 - 595）和马铃薯phyB 66 kDa（1 - 596）的N端脱辅基蛋白片段在大肠杆菌以及酿酒酵母和毕赤酵母中进行了异源表达，并与藻胆素（PthetaB；天然发色团）和藻蓝胆素（PCB）组装。来自酵母的phyA65脱辅基蛋白在最初的急剧上升后呈现单指数组装动力学，而来自大肠杆菌的相应脱辅基蛋白仅呈现缓慢的单指数组装。phyB66脱辅基蛋白掺入任何一种发色团的速度都比phyA65慢，具有双指数动力学。对于所有脱辅基蛋白，PthetaB的掺入速度都比PCB快。N端半段的Pfr形式的热稳定性与全长重组光敏色素已知的热稳定性相似：燕麦phyA65 Pfr高度稳定，而马铃薯phyB66 Pfr迅速转化为Pr。因此，C端结构域和同二聚体形成均不调节此特性。相反，这是光敏色素的一个特征，表明其源自单子叶植物或双子叶植物。N端phyA65和phyB66从Pr到Pfr的动力学不同。phyA65 - PCB的初级光产物I700呈单指数衰减，而PthetaB类似物呈双指数衰减，而phyB66的I700无论掺入何种发色团均呈单指数衰减。N端半段从Pr形成Pfr的速度比全长光敏色素快，表明C端结构域参与了相对缓慢的蛋白质构象变化。
